Improved synaptic and cognitive function in aged 3 × Tg-AD mice with reduced amyloid-ß after immunotherapy with a novel recombinant 6Aß15-TF chimeric vaccine.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder impairing memory and cognition. In this study, we describe the immunogenicity and protective efficacy of the novel recombinant 6Aß15-TF chimeric antigen as a subunit protein vaccine for AD. Recombinant 6Aß15-TF chimeric vaccine induced strong Aß-specific humoral immune responses without Aß-specific T cell immunity in C57/BL6 and 3 × Tg-AD mice at different ages. As an early immunotherapy model for AD, this vaccine induced high titers of long-lasting anti-Aß42 antibodies in aged 3 × Tg-AD mice, which led to improve behavioral performance and markedly reduced the levels of insoluble and soluble Aß and Aß oligomers. In agreement with these findings, immunotherapy with 6Aß15-TF prevented the Aß-induced decrease of presynaptic and postsynaptic proteins in aged 3 × Tg-AD mice. Our results suggest that this novel and highly immunogenic recombinant 6Aß15-TF chimeric vaccine provides neuroprotection in AD mice and can be considered an effective AD candidate vaccine.

Enhanced effects of DNA vaccine against botulinum neurotoxin serotype A by targeting antigen to dendritic cells.

As dendritic cells (DCs) play a critical role in priming antigen-specific immune responses, the efficacy of DNA vaccines may be enhanced by targeting the encoded antigen proteins to DCs. In this study, we constructed a DC-targeted DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A (AHc) fused with scDEC, a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Intramuscular injections of mice with the DC-targeted DNA vaccine (pVAX1-scDEC-AHc) stimulated more DCs to mature than the non-targeted DNA vaccine (pVAX1-SAHc) in the splenocytes. The DC-targeted DNA vaccine could induce more DCs maturation at the site of inoculation. The DC-targeted DNA vaccine induced stronger AHc-specific humoral immune responses, lymphocyte proliferative responses and protective potency against BoNT/A in mice than did pVAX1-SAHc. Moreover, the DC-targeting DNA vaccine provided effective protection after only two inoculations. In summary, these results showed that the DC-targeted fusion DNA vaccine could generate strong immunity, indicating that maturation of DCs induced by pVAX1-scDEC-AHc may be helpful for priming and boosting immune responses. Thus, we propose that the strategy of targeting antigen to DCs in vivo via DEC205 can enhance effectively the potency of DNA vaccines against BoNTs or other pathogens in an animal model.

The highly pathogenic H5N1 influenza A virus down-regulated several cellular MicroRNAs which target viral genome.

Higher and prolonged viral replication is critical for the increased pathogenesis of the highly pathogenic avian influenza (HPAI) subtype of H5N1 influenza A virus (IAV) over the lowly pathogenic H1N1 IAV strain. Recent studies highlighted the considerable roles of cellular miRNAs in host defence against viral infection. In this report, using a 3'UTR reporter system, we identified several putative miRNA target sites buried in the H5N1 virus genome. We found two miRNAs, miR-584-5p and miR-1249, that matched with the PB2 binding sequence. Moreover, we showed that these miRNAs dramatically down-regulated PB2 expression, and inhibited replication of H5N1 and H1N1 IAVs in A549 cells. Intriguingly, these miRNAs expression was differently regulated in A549 cells infected with the H5N1 and H1N1 viruses. Furthermore, transfection of miR-1249 inhibitor enhanced the PB2 expression and promoted the replication of H5N1 and H1N1 IAVs. These results suggest that H5N1 virus may have evolved a mechanism to escape host-mediated inhibition of viral replication through down-regulation of cellular miRNAs, which target its viral genome.

A novel recombinant 6Aß15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer's disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-ß (Aß) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aß15-THc-C immunogen was formulated with alum adjuvant as a novel Aß B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aß-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aß or oligomeric forms of Aß, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aß levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aß-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine.

A Novel Aß B-Cell Epitope Vaccine (rCV01) for Alzheimer's Disease Improved Synaptic and Cognitive Functions in 3 × Tg-AD Mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive amyloid-ß accumulation, loss of cognitive abilities, and synaptic alterations. Given the remarkable recovery of cognition in AD models of targeting-Aß immunotherapy, we sought to determine the molecular correlate(s) associated with improvement. We evaluated the efficacy of a recombinant chimeric 6Aß15-T antigen formulated with alum adjuvant as a novel Aß B-cell epitope vaccine (rCV01) in 3 × Tg-AD mice. rCV01 elicited robust Th2-polarized Aß-specific antibodies without autoimmune T cell responses in 3 × Tg-AD mice. The long-lasting anti-Aß42 antibodies were associated with markedly reduced AD-like pathology, enhanced synaptic function, and improved cognitive performance in aged 3 × Tg-AD mice. This is the first report to provide one hypothesis for the improved outcomes following vaccination is a reduction in the levels of active calpain in rCV01-immunized AD mice, which is likely attributable to preventing dynamin 1 and PSD-95 degradation allowing functional recovery of cognition. rCV01 is a highly immunogenic recombinant chimeric 6Aß15-T vaccine that shows clear neuroprotective properties in preclinical mouse models of AD and is a candidate for an effective AD vaccine.

Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells.

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. However, the exact mechanism of EZH2 up-regulation in HCC has not been determined. In this study, we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein (HBx) in regulating the expression of mEZH2. Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner. To further investigate the mechanism of mEZH2 overexpression, the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid. The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid, and deleting the -486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%. The -486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

Development and evaluation of candidate vaccine and antitoxin against botulinum neurotoxin serotype F.

To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype F (rFHc) was expressed in Escherichia coli and purified by sequential chromatography. The rFHc was evaluated as a subunit vaccine candidate in mouse model of botulism. A dose-response was observed in both antibody titer and protective efficacy with increasing dosage of rFHc and number of vaccinations. These findings suggest that the rFHc is an effective botulism vaccine candidate. Further, we developed a new antitoxin against botulinum neurotoxin serotype F (BoNT/F) by purifying F(ab')(2) fragments from pepsin digested serum IgGs of horses inoculated with rFHc. The protective effect of the F(ab')(2) antitoxin against BoNT/F was determined both in vitro and in vivo. The results showed that the F(ab')(2) antitoxin could prevent botulism in mice challenged with BoNT/F and effectively delayed progression of paralysis from botulism in the therapeutic setting. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/F.

Development and preclinical evaluation of a new F(ab')2 antitoxin against botulinum neurotoxin serotype A.

Concern about the malicious applications of botulinum neurotoxin has highlighted the need for a new generation of safe and highly potent antitoxins. In this study, we developed and evaluated the preclinical pharmacology and safety of a new F(ab')2 antitoxin against botulinum neurotoxin serotype A (BoNT/A). As an alternative to formalin-inactivated toxoid, the recombinant Hc domain of botulinum neurotoxin serotype A (rAHc) was used to immunize horses, and the IgGs from the hyperimmune sera were digested to obtain F(ab')2 antitoxin. The protective effect of the new F(ab')2 antitoxin against BoNT/A was determined both in vitro and in vivo. The results showed that the F(ab')2 antitoxin could prevent botulism in mice challenged with BoNT/A and effectively delayed progression of paralysis from botulism in the therapeutic setting. The preclinical safety of the new F(ab')2 antitoxin was also evaluated, and it showed neither harmful effects on vital functions nor adverse effects such as acute toxicity, or immunological reactions in mice and dogs. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/A, and our findings support the safety of the new F(ab')2 antitoxin for clinical use. Our study further demonstrates the proof of concept for development of a similar strategy for obtaining potent antitoxin against other BoNT serotypes.

Spinal antinociception of synthetic omega-conotoxin SO-3, a selective N-type neuronal voltage-sensitive calcium channel blocker, and its effects on morphine analgesia in chemical stimulus tests in rodent.

SO-3, a novel Omega-superfamily conotoxin derived from Conus striatus, selectively inhibits N-type neuronal voltage-sensitive calcium channels. In current study, antinociception of SO-3 compared with MVIIA or morphine and its effects on morphine analgesia were investigated in rodent chemical stimulus tests after acute or repeated intrathecal administration. In mice acetic acid writhing test, similar to MVIIA, SO-3 caused dose- and time-dependent spinal antinociception with ED(50) of 0.25 microg/kg and t(1/2) of 4h, which was more potent and longer-acting than morphine. In rat formalin test after intrathecal bolus injection, SO-3 produced dose- and time-dependent antinociception by suppressing acute (ED(50), 1.79 microg/kg) and tonic phases (ED(50), 0.41 microg/kg), which was similar to MVIIA and approximately 10-fold potency and twice longer-acting of morphine in blocking tonic phase responses. After repeated intrathecal injections twice daily for 5 consecutive days, SO-3 produced analgesia without loss of potency whereas morphine produced analgesia tolerance in rat formalin test; further, SO-3 still produced potent analgesia in morphine-tolerant rats. SO-3 co-administered with morphine left-shift the dose-response curve of morphine in mice acetic acid writhing test and significantly potentiated morphine analgesia in rat formalin test. No changes in motor function were seen in mice or rats receiving antinociceptive doses of SO-3 whereas MVIIA caused motor dysfunction at doses of 1.0-2.0 microg/kg in rats. This study showed that (1) novel SO-3 produced potent and long-acting spinal antinociception without observable motor dysfunction, (2) SO-3 significantly potentiated morphine analgesia, (3) After repeated intrathecal administration, SO-3 produced neither tolerance nor cross-tolerance to morphine analgesia.

Silencing PinX1 compromises telomere length maintenance as well as tumorigenicity in telomerase-positive human cancer cells.

The nucleolar protein PinX1 has been proposed to be a putative tumor suppressor due to its binding to and inhibition of the catalytic activity of telomerase, an enzyme that is highly expressed in most human cancers in which it counteracts telomere shortening-induced senescence to confer cancer cell immortalization. However, the role of PinX1 in telomere regulation, as well as in cancer, is still poorly understood. In this study, we showed that the PinX1 protein is constitutively expressed in various human cells regardless of their telomerase activity and malignant status. Most interestingly, we found that silencing PinX1 expression by a potent short hairpin RNA construct led to a robust telomere length shortening and growth inhibition in telomerase-positive but not in telomerase-negative human cancer cells. We further showed that silencing PinX1 significantly reduced the endogenous association of telomerase with the Pot1-containing telomeric protein complex, and therefore, could account for the phenotypic telomere shortening in the affected telomerase-positive cancer cells. Our results thus reveal a novel positive role for PinX1 in telomerase/telomere regulations and suggest that the constitutive expression of PinX1 attributes to telomere maintenance by telomerase and tumorigenicity in cancer cells.

Nucleolar localization of TERT is unrelated to telomerase function in human cells.

Telomerase maintains telomere length and has been implicated in both aging and carcinogenesis of human cells. This enzyme is a specialized ribonucleoprotein (RNP) complex, minimally consisting of two essential components: the protein catalytic subunit TERT (telomerase reverse transcriptase) and the integral RNA moiety TR (telomerase RNA, TERC). Both TERT and TR have been found to localize to nucleoli within the nucleus, leading to the suggestion of nucleoli as the site for telomerase RNP biogenesis in human cells. However, whether this statement is true or not has not yet been convincingly demonstrated. Here, we identify that residues 965-981 of the human TERT polypeptide constitute an active nucleolar-targeting signal (NTS) essential for mediating human TERT nucleolar localization. Mutational inactivation of this NTS completely disrupted TERT nucleolar translocation in both normal and malignant human cells. Most interestingly, such a TERT mutant still retained the capacity to activate telomerase activity, maintain telomere length and extend the life-span of cellular proliferation, as does wild-type TERT, in BJ cells (normal fibroblasts). Therefore, our data suggest that TERT nucleolar localization is unrelated to telomerase function in human cells.

Effect of O-superfamily conotoxin SO3 on synchronized spontaneous calcium spikes in cultured hippocampal networks.

SO3 belongs to the O-superfamily of conotoxins and is known to have analgesic effects in experimental animals. In order to explore the mechanism of its potential pharmacological actions, the effect of SO3 on synchronized spontaneous calcium spikes was examined in cultured hippocampal networks by calcium imaging. Spontaneous oscillations of intracellular concentrations of calcium (Ca(2+)) in the form of waves and spikes are found in cultured hippocampal networks. Exposure to increasing concentrations of SO3 resulted in a progressive decrease in synchronized spontaneous calcium spikes. The higher concentrations (0.1 micromol/L and 1 micromol/L) of SO3 showed the strongest inhibition. The rank order of inhibition was 1 micromol/L > 0.1 micromol/L > 10 micromol/L > 0.01 micromol/L. This action of SO3 in reducing synchronized calcium spikes suggests a possible application for therapeutic treatment of epilepsy.

[Cloning NS1 gene of H5N1 avian influenza virus and apoptosis induced by it in human pulmonary carcinoma cell line A549].

The NS1 gene of the H5N1 subtype avian influenza virus was amplified by RT-PCR, and the am-plified product was cloned into the eukaryotic expression vector pCMV-Myc, then it was transfected into A549 cells. After 48 h, the expression of NS1 was detected by Western blot. Fluorescence and electron microscopy and flow cytometry showed that the NS1 gene of H5N1 avian influenza virus could induce apop-tosis in human pulmonary carcinoma cell line A549.

[Establishment of osteoblast-specific Cre transgenic mice].

An osteoblast-specific Cre transgenic construct (pOC-Cre) containing the osteocalcin promoter, Cre recombinase gene and polyA of human growth hormone gene was generated. The 4.6 kb DNA fragment of OC-Cre was introduced into fertilized zygotes by microinjection. 2 out of 16 offspring were identified as founders carrying the transgene by PCR and Southern blot analysis, and the integration efficiency is 12.5 %. To check the tissue distribution of the OC-Cre, the OC-Cre transgenic founder mice were bred with the mice carrying Smad4 conditional alleles. PCR results showed that the genomic DNA fragments after Cre mediated recombination could be only amplified from bone tissues of the transgenic mice. LacZ staining of OC-Cre; ROSA26 double transgenic mice revealed that Cre recombinase expressed in osteoblasts and mediated DNA recombination between LoxP sites at the ROSA26 locus. All these data indicated that the Cre recombinase was ex-pressed in the osteoblasts of the OC-Cre transgenic mice and could mediate DNA recombination between LoxP sites. The OC-Cre transgenic mice we generated in this study could serve as a useful tool for generating osteoblast specific gene-knockout mice.

Dynamic proteome changes of Shigella flexneri 2a during transition from exponential growth to stationary phase.

Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD (OmpW) was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced (such as AnsB, AspA, FrdAB, and KatG) or repressed (such as AceEF, OmpX, SodA, and SucAB) during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.

PC-1/PrLZ contributes to malignant progression in prostate cancer.

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.

Identification and characterization of the novel human prostate cancer-specific PC-1 gene promoter.

Human prostate and colon gene-1 (PC-1, also known as PrLZ) is an androgen-regulated, prostate tissue and prostate cancer cells specifically expressed novel gene. The increased expression of PC-1 gene appears to promote prostate cancer cells androgen-dependent (AD) and androgen-independent (AI) growth. To clone and investigate the expression and regulation elements of PC-1 gene may provide insight into the function of PC-1 and develop a new promoter that targets therapeutic genes to the AD and AI prostate cancer cells. The goal of the present study is cloning and characterization of the PC-1 promoter. A series of luciferase constructs that contain various fragments of the PC-1 5'-genomic region were transfected into human prostate cancer cells for promoter transactivation analysis. 5' deletion analysis identified the -1579 bp promoter region was required for the maximal proximal promoter activity; two transcriptional suppression and a positive regulatory region were identified; -4939 bp promoter fragment of the PC-1 gene retained the characteristic of prostate cancer-specific expression and exhibited higher transcription activity than PSA-6 kb promoter in the medium supplemented with steroid-depleted FBS. An androgen response element (ARE) was located in between -345 and -359 bp of the PC-1 5'-untranslated region relative to the translation initiation site. Thus, our studies not only provide molecular basis of PC-1 transcription regulation, but also define a new regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer in the prostate cancer gene therapy.

[The targeting study of human serum albumin gene].

To generate transgenic porcine which expresses human serum albumin (HSA), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2 kb 5' regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3' end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.

[Enhancement of exogenous gene expression by artificial transcription factor in CHO cells].

Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA-binding domain and four tandem repeats of the 12-aa peptide (DALDDFDLDMLG) of the herpesvirus as the activation domain, an artificial transcription factor, GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40. And then, GVP4 was cloned into expression vector pcDNA3 . 1/Hygro ( + ) . Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1 ( + ) that separately harbored EGFP cDNA and t-PA cDNA.The CHO cells were then co-transfected with GVP4 expression vector and EGFP or t-PA expression vector. The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t-PA in the supernatant. GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor. And, CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression, which resulted in 2-3-fold increase in both EGFP and t-PA expressions. These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.

[Design and implementation of DB sequence optimization software].

TIR (Translation Initiation Region) efficiency is very important in prokaryotic expression. The TIR's efficiency is highly dependent on SD (Shine-Dalgarno) sequence, distance between SD sequence and start codon, DB (Downstream Box) sequence, TIR's second structure, codon adaptation and so on. In this paper, we designed and implemented the software to optimize DB sequence and 5' rare codons. It generated some optimization sequences by analyzing the target sequence and comparing it with 16S RNA. And the optimization sequences is sorting by number of base pairing, location of base pairing and codon adaptation. We drew up the algorithm and the core of code in this paper.